Soybean protein has been widely utilized for improving physical properties of foods because of its peculiar gelling property, and at the same time, use of soybean protein as a highly nutritious health food material has been increased.
A storage protein of a soybean is precipitated at around pH 4.5, and therefore soybean protein can be relatively simply separated into an acid-soluble protein fraction mainly containing soluble components other than a storage protein and an acid-precipitable protein fraction mainly containing a storage protein. The acid-precipitable protein fraction is collected to obtain an isolated soybean protein, which has been currently widely used in the food industry.
Proteins constituting a soybean protein are classified into 2S globulin, 7S globulin, 11S globulin and 15S globulin based on sedimentation coefficient by ultracentrifuge analysis. Among them, 7S globulin and 11S globulin are the main constituent protein components of a globulin fraction. Herein, β-conglycinin and glycinin according to immunological nomenclature substantially correspond to 7S globulin and 11S globulin respectively.
Proteins constituting soybean protein are each different in physical properties such as viscosity, coagulation property and surface activity, and nutritional physiological functions.
For example, it has been reported that 7S globulin decrease neutral fat in blood (see Non-Patent Document 1). It has been believed that 11S globulin has a high gelling activity and governs the hardness and mouth feeling of a bean curd gel.
Thus, fractionation of soybean protein into fractions rich in these components enables physiological functions or physical properties peculiar to each protein component to greatly manifest, probably leading to creation of a characteristic material. Therefore, extension of the field utilizing soybean protein in the food industry can be expected.
As seen from FIG. 1 showing dissolution behavior of 7S globulin and 11S globulin for a pH, 7S globulin has a low solubility at about pH 4.8 and 11S globulin has a low solubility at from pH 4.5 to 6. Thus, it is expected that respective components with a high purity may be fractionated by first precipitating 11S globulin at about pH 6 and then further lowering pH to precipitate 7S globulin.
However, in fact, when soybean milk is adjusted to pH 6 and separated into an insoluble fraction and a water-soluble fraction and then, these fractions are subjected to SDS-polyacrylamide gel electrophoresis, the obtained electrophoresis patterns show that considerable amounts of 7S globulin and 11S globulin are mixed in both fractions.
For this reason, there is a problem that only a simple fractionation based on dissolution behavior of both globulins for a pH can not obtain their fractions with a high purity.
In order to overcome such a problem, some techniques for fractionation of 7S globulin and 11S globulin have been disclosed (see Non-Patent Document 2, Patent Documents 1 to 7 etc.).
On the other hand, it has been reported in recent years that an acid-precipitable soybean protein fraction comprises various proteins having high affinity for polar lipids which constitutes the membrane of a protein body, an oil body and the like including a cell membrane, in addition to 7S globulin and 11S globulin (see Non-Patent Document 3).
Considering such a report, the present inventors studied. As a result, they found that when sodium sulfate was added to low-denatured defatted soybean milk so as to be a 1 M concentration and the soybean milk was then adjusted to pH 4.5 with hydrochloric acid, 7S and 11S globulins transferred into an acid-soluble fraction and other various proteins transferred into an acid-precipitable fraction (see Non-Patent Document 4).
It was also found that the nitrogen amount of the acid-precipitable fraction accounted for about 30% of the total nitrogen amount in the defatted soybean milk, which was an unexpected large amount.
Further, it has been reported that an industrially produced isolated soybean protein contains about 35% of these various proteins, and it has been found that such a group of proteins influence the flavor of a conventional soybean protein material such as soybean milk or an isolated soybean protein (see Non-Patent Document 5).
The acid-precipitable fraction that is not rich in 7S globulin and 11S globulin comprises proteins having mainly a deduced molecular weight of 34 kDa, 24 kDa and 18 kDa based on SDS-polyacrylamide electrophoresis, lipoxygenase, γ-conglycinin and other many various proteins. Such a group of proteins have affinity for a polar lipid.
According to the above described findings, it is understood that the previous fractionation techniques (Non-Patent Document 2, Patent Documents 1 to 7) could not substantially attain fractionation of 7S globulin and 11S globulin with a high purity because it was not previously considered at all that lipophilic proteins account for a considerable proportion of an acid-precipitable soybean protein fraction.
Although Non-Patent Document 4 has shown a method for fractionation of 7S globulin, 11S globulin and a lipophilic protein with a high purity, said method needs use of a large amount of a reducing agent under a high ionic strength and thus needs a desalting step and a washing step. Therefore, said method is effective at the experimental level, but is not suitable for an industrial process.
Then, the present applicant developed a technique for fractionating a soybean protein into a soybean 7S globulin protein fraction with a high purity which has a low content rate of a lipophilic protein, and a soybean 11S globulin protein fraction (see Patent Documents 8 and 9). Said method was industrially excellent in that 7S globulin with a high purity could be fractionated. However, in order to obtain a soybean 11S globulin fraction with a high purity which has a decreased content rate of a lipophilic protein, a troublesome procedure was needed. Therefore there was room for improvement in said method.
In other words, development of a process for preparing a general isolated soybean protein or both of 7S globulin and 11S globulin with a low content rate of a lipophilic protein, but not a process for fractionating only 7S globulin with a high purity, is desired. In addition, a simple method which can fractionate each of 7S globulin, 11S globulin and a lipophilic protein with a high purity is desired.